The cell cycle for growing cells can be divided into two periods: (1) the cell division period, when the cell divides and separates, with each daughter cell receiving identical copies of the DNA; and (2) the period of growth, known as the interphase period. For the cell cycle of eucaryotes, the cell division period is labeled the M (mitotic) period. The interphase period in eucaryotes is further divided into three successive phases: G1 (gap 1) phase, which directly follows the M period; S (synthetic) phase, which follows G1; and G2 (gap 2) phase, which follows the S phase, and immediately precedes the M period. During the two gap phases no net change in DNA occurs, though damaged DNA may be repaired. On the other hand, throughout the interphase period there is continued cellular growth and continued synthesis of other cellular components. Towards the end of the G1 phase, the cell passes a restrictive (R) point and becomes committed to duplicate its DNA. At this point, the cell is also committed to divide. During the S phase, the cell replicates DNA. The net result is that during the G2 phase, the cell contains two copies of all of the DNA present in the G1 phase. During the subsequent M period, the cells divide with each daughter cell receiving identical copies of the DNA. Each daughter cell starts the next round of the growth cycle by entering the G1 phase.
The G1 phase represents the interval in which cells respond maximally to extracellular signals, including mitogens, anti-proliferative factors, matrix adhesive substances, and intercellular contacts. Passage through the R point late in G1 phase defines the time at which cells lose their dependency on mitogenic growth factors for their subsequent passage through the cycle and, conversely, become insensitive to anti-proliferative signals induced by compounds such as transforming growth factor, cyclic AMP analogs, and rapamycin. Once past the R point, cells become committed to duplicating their DNA and undergoing mitosis, as noted above, and the programs governing these processes are largely cell autonomous.
In mammalian cells, a molecular event that temporally coincides with passage through the R point is the phosphorylation of the retinoblastoma protein (RB). In its hypophosphorylated state, RB prevents the cell from exiting the G1 phase by combining with transcription factors such as E2F to actively repress transcription from promoters containing E2F binding sites. However, hyperphosphorylation of RB late in G1 phase prevents its interaction with E2F, thus allowing E2F to activate transcription of the same target genes. As many E2F-regulated genes encode proteins that are essential for DNA synthesis, RB phosphorylation at the R point helps convert cells to a pre-replicative state that anticipates the actual G1/S transition by several hours. Cells that completely lack the RB function have a reduced dependency on mitogens but remain growth factor-dependent, indicating that cancellation of the RB function is not sufficient for passage through the R point.
Phosphorylation of RB at the R point is initially triggered by holoenzymes composed of regulatory D-type cyclin subunits and their associated cyclin-dependent kinases, CDK4 and CDK6. The D-type cyclins are induced and assembled into holoenzymes as cells enter the cycle in response to mitogenic stimulation. Acting as growth factor sensors, they are continuously synthesized as long as mitogenic stimulation continues, and are rapidly degraded after mitogens are withdrawn. In fibroblasts, inhibition of cyclin D-dependent CDK activity prior to the R point, either by microinjection or by scrape loading of antibodies directed against cyclin D1 or by expression of CDK4 and CDK6 inhibitors (INK4 proteins) prevents entry into S phase. However, such manipulations have no effect in cells lacking functional RB, implying that RB is the only substrate of the cyclin D-dependent kinases whose phosphorylation is necessary for exiting the G1 phase.
Since RB-mediated controls are not essential to the cell cycle per se it is difficult to understand why mammalian cells contain three distinct D-type cyclins (D1, D2, and D3), at least two cyclin D-dependent kinases (CDK4 and CDK6), and four INK4 proteins, all, purportedly, for the sole purpose of regulating RB phosphorylation. This apparent redundancy has been explained as a method to govern transitions through the R point in different cell types responding to a plethora of distinct extracellular signals.
Alternatively, cyclin D-dependent kinases, or the cyclins alone could also be involved in the regulation of RB-independent events, perhaps linking them temporally to cell cycle controls. One mechanism for this regulation could involve the direct interaction between a cyclin, such as a D-type cyclin, and a specific transcription factor, which would allow the cyclins to regulate gene expression in an RB-independent manner. However, up until now, no such RB-independent transcription factor has been identified.
The citation of any reference herein should not be deemed as an admission that such reference is available as prior art to the instant invention.